Radioimaging and radiotherapy utilize cell or tissue specific targeting agents as delivery systems for radioactive, paramagnetic or cytotoxic agents. Any agent which is specific for a lesion or site of interest can potentially act as a targeting agent. For example, polyclonal and monoclonal antibodies can be produced which exhibit considerable specificity for certain cell or tissue types. Many other agents, including toxins such as diphtheria toxin, exhibit cell specificity and can be used to deliver diagnostic or therapeutic agents. The technique of delivery of monoclonal antibodies (MAbs) has been investigated for cancer therapy as well as for diagnosis of cancer, thromboembolism and cardiac myopathy. For successful radioimmunoimaging, sufficient labeled MAb must localize at the target site to provide enough signal for detection. Target-to-background ratios must be high in order to achieve adequate contrast between target-bound radioactivity and background levels in other organs, tissues and blood. A major obstacle to successful radioimmunoimaging is the high background activity of free circulating radiolabeled MAbs due to prolonged circulation and accumulation in liver and spleen, the normal metabolic sites for Abs. Furthermore, the toxic effects of high radiation doses must be considered in both radioimmunotherapy and radioimmunoimaging. Such obstacles are also a consideration for methods utilizing targeting agents other than monoclonal antibodies.
To overcome such obstacles, "pre-targeting" or "two-step" approaches have been investigated. In the conventional one-step method the radionuclide is linked to the MAb either directly or via a bifunctional chelating agent. In the two-step approach the antibody is unlabeled. Unlabeled antibody is administered, and antibody which does not localize to the target site is allowed to clear from circulation before the administration of radioactivity. The radioactivity is then administered in a chemical form which has high affinity for the antibody.
To provide the diagnostic or imaging agent in a form with high affinity for the antibody, two-step methods have been designed to exploit the high affinity of avidin and streptavidin for biotin. Avidin, a 67 kilodalton (kD) glycoprotein found in egg whites, has an exceptionally high binding affinity (K.sub.a =10.sup.-15) for biotin. Avidin consists of four subunits, each capable of binding one biotin molecule. Streptavidin, a similar protein produced in Streptomyces avidinii, shares significant conformation and amino acid composition with avidin, as well as high affinity and stability for biotin. However, streptavidin is not glycosylated and reportedly exhibits less non-specific binding to tissues. Streptavidin is widely used in place of avidin because of its lower non-specific binding. Biotin, a member of the B-complex vitamins, is essential for amino acid and odd-chain fatty acid degradation, gluconeogenesis and fatty acid synthesis and is normally found in the enzyme bound form as biocytin.
The use of the two-step avidin-biotin or streptavidin-biotin approach for radioimmunoimaging and radioimmunotherapy is theoretically attractive since: 1) biotin and avidin are likely to be nontoxic at the levels required for these applications; 2) the high affinity of avidin and biotin results in the in vivo stability of the avidin-biotin bond; 3) the rapid clearance of biotin through the kidney avoids problems associated with use of radiolabeled MAbs; and 4) the tetravalency of avidin and streptavidin for biotin allows for amplification of the signal at the target site.
In the two-step avidin-biotin or streptavidinbiotin approach, antibodies are coupled with either biotin or avidin and administered to the subject, followed by administration of radiolabeled avidin or biotin, respectively. Using an animal non-tumor model, Hnatowich et al. [(1987) J. Nucl. Med. 28, 1294] have demonstrated the administration of avidin-conjugated antibody to mice, followed by administration of .sup.111 In-labeled biotin. Imaging was performed to determine the ratio of radioactivity in the target organ relative to other organs. Using Protein A-conjugated beads to simulate tumor, Hnatowich et al. thus demonstrated localization of the label to the target, with improved target-to-nontarget ratios relative to the conventional one-step approach. In a similar study, Paganelli et al. [(1988) Int. J. Cancer 2, 121] demonstrated the in vivo labeling of biotinylated antibody with .sup.131 I-and .sup.111 In-labeled avidin.
Kalofonos et al. [(1990) J. Nucl. Med. 31, 1791] have reported preliminary results of a limited clinical trial in which patients with squamous cell carcinoma of the lungs received streptavidin-conjugated monoclonal antibody followed by .sup.111 In-labeled biotin. In eight out of ten patients, tumor was detected with labeled biotin alone without the previous administration of streptavidin-conjugated antibody, perhaps due to localization of labeled biotin in tumor. In three of these patients, images were improved with the prior administration of antibody. The fact that targeting was not improved in all patients was speculated to be due to rapid internalization of antibody but may be the result of uptake of biotin by tumor cells.
Clinical studies demonstrating a two-step approach (biotinylated MAb followed by .sup.111 In-labeled streptavidin) and a three-step approach (biotinylated MAb followed by cold avidin followed by .sup.111 In-labeled biotin) have also been reported by Paganelli et al. [(1990) J. Nucl. Med. 31, 735].
In the above studies, biotin, avidin and streptavidin were each labeled with .sup.111 In via the bifunctional chelating agent diethylentriaminepentaacetic acid (DTPA) by the bicyclic anhydride method of Hnatovich et al. [(1982) Int. J. Appl. Radiat. Isotop. 33, 327].
A bifunctional chelating agent is a reagent which has the ability to bind to a metal ion as well as to bind to a protein or antibody. The use of chelating agents to radiolabel biotin has advantages over direct labeling since the resulting complexes are more likely to be stable and to retain biological activity. However, the use of DTPA as a chelating agent to label biotin presents significant limitations to the development of two-step radioimmunoimaging and radioimmunotherapy. DTPA-biotin is a dimer containing two biotin molecules (biotin-DTPA-biotin). Since each molecule of avidin binds four biotin molecules, a maximum of two molecules of DTPA-biotin can bind per mole of avidin or avidin-conjugated antibody, thus limiting the amount of radioactivity that can be delivered to the target site.
Another disadvantage of DTPA-biotin as a labeling reagent is a consequence of the conjugation of DTPA and biotin. DTPA-biotin is prepared by covalently linking DTPA to biotin through the use of the cyclic anhydride of DTPA and biocytin, a lysine conjugate of biotin with an available primary amine for conjugation (Hnatowich, 1987). DTPA is a member of the aminocarboxylic acid class of chelating agents, having five deprotonated carboxylate groups and three tertiary amino groups for binding to the metal ion. Since two of the five COO.sup.- groups are used for conjugation to biocytin, DTPA is rendered hexadentate rather than octadentate, leading to decreased thermodynamic stability and consequent label instability. Maecke et al. [(1989) J. Nucl. Med. 30, 1235] have demonstrated that the octadentate ligand of .sup.111 In-DTPA is superior to ligands with smaller denticity for in vivo applications.
A further disadvantage of DTPA as a chelating agent is its affinity for divalent cations found in vivo, especially Mg.sup.2+ and Ca.sup.2+ (stability constants of 9 and 11, respectively). The affinity of DTPA for divalent cations allows for the exchange of the radiometal with divalent metal in in vivo applications. For example, In and Y are released from DTPA-MAbs at a rate of about 10 percent per day in vivo [Hnatowich et al., (1985) J. Nucl. Med. 26, 503], and would be replaced by Ca.sup.2+ and Mg.sup.2+, which are present in much higher concentrations. In addition to reducing the amount of radiometal delivered to the target site, a further problem is created by the exchange of radiometal for divalent cation. Certain released radiometals will become bound by transferrin, a human plasma metal binding protein. For example, the affinity of transferrin for Fe and Ga is higher than the affinity of DTPA for these metals, thus promoting the leaching of Fe and Ga off of DTPA and onto transferrin. The consequence of this leaching is the eventual sequestration of the radiometal-transferrin complex to the liver and bone marrow, a tissue which is particularly susceptible to radiation damage.
Another consequence of the affinity of DTPA for divalent cations is a reduction in labeling efficiency due to the presence of these competing impurities in radionuclide solutions.
A still further disadvantage of DTPA occurs because the affinity of DTPA for metal ions is pH sensitive. Since the deprotonated form is the chelating moiety, metal binding affinity is decreased at low pH, a condition which may be present in tumors and abscesses. Brechbiel et al. [(1986) Inorgan. Chem. 25, 2772] and Meares [(1986) Nucl. Med. Biol. 13, 311], have reported that anionic complexes of DTPA with Cu, In or Y are susceptible to acid- or cation-catalyzed dissociation, and that free metal ions are prematurely released in vivo with a propensity to accumulate in liver, bone and bone marrow.
Accordingly, a need exists for better biotin conjugates which can be labeled with radionuclides suitable for diagnosis and therapy. The present invention provides conjugates of deferoxamine and biotin which overcome the deficiencies of the compounds of the prior art.
Deferoxamine (DFO) is a trivalent metal chelating agent with extremely high affinity for transition metals [e.g. K.sub.d =10.sup.-30 M.sup.-1 for iron(III)]. It contains three hydroxamic groups and reacts stoichiometrically with tripositive metal ions to form octahedral complexes. Deferoxamine mesylate is commercially available and has been established as a safe drug for the short-term treatment of iron toxicity, iron storage disease and iron and aluminum overload. Due to its high affinity for other metals, DFO is potentially useful in certain modalities of radiology, including nuclear medicine and magnetic resonance imaging, when bound to radioactive metals such as .sup.99m Tc, .sup.111 In, .sup.67 Ga, .sup.90 Y, .sup.186 Re, .sup.18 Re, .sup.212 Bi, or paramagnetic ions such as Fe and Gd. DFO forms stable metal chelates, and has a reactive amino group available for coupling with proteins.
DFO forms an uncharged chelate of compact structure, thus minimizing its effect on the biological properties of the protein to which it is conjugated. DFO has been used to prepare .sup.67 Ga-labeled human serum albumin [Yokoyama et al. (1981) J. Nucl. Med. 23, 909] and .sup.67 Ga-labeled fibrinogen [Ohmomo et al. (1982) Eur. J. Nucl. Med. 7, 458]. Takahashi et al. [(1985) Viena IAEA 471], provide a method for increasing the specific activity of .sup.67 Ga-labeled fibrinogen by coupling DFO to fibrinogen through a functional polymer, dialdehyde starch (DAS), to provide a .sup.67 Ga-fibrinogen-(DAS-DFO) conjugate. Yamamoto et al. [(1988) Eur.J. Nucl. Med. 14, 60], have evaluated .sup.67 Ga-fibrinogen-(DAS-DFO) for imaging of venous thrombi. U.S. Pat. No. 4,680,338 to Sundoro discloses a bifunctional sequential linker and its use to minimize crosslinking of amine ligands, and contemplates its use in the preparation of a DFO-antibody conjugate. Kubodera et al. (U.S. Pat. No. 4,888,163) and Yazaki et al. (U.S. Pat. No. 4,943,427) also contemplate the preparation of a radiolabeled DFO-antibody conjugate.
DFO provides significant advantages over DTPA as a bifunctional chelating agent for two-step radioimmunoimaging and radioimmunotherapy. DFO has one primary amine available for coupling reactions with proteins, and thus a DFO-biotin conjugate would be expected to be a monomer, i.e. one molecule of DFO per molecule of biotin. Accordingly, a DFO-biotin conjugate has the potential to deliver twice the molar amount of metal per biotin molecule relative to a, dimer of DTPA-biotin. Furthermore, the amine group available for conjugation to biotin is spaced by five carbon atoms from the nearest hydroxamic group involved in chelation. Thus, in contrast to DTPA-biotin, conjugation of biotin to DFO should not appreciably affect the affinity of DFO for metal ions.
Another advantage of DFO as a bifunctional chelating agent is its low affinity for divalent metals. The stability constants of Ca.sup.2+ and Mg.sup.2+ with DFO are 3 and 4, respectively. Accordingly, exchange of radiometal with Ca.sup.2+ and Mg.sup.2+, which are present in high concentrations in body fluids and buffers, is minimal.
Weiner et al. [(1979) in Proceedings of the Second International Symposium on Radiopharmaceuticals, 331], have demonstrated that a .sup.67 Ga-DFO complex is much stronger than a .sup.67 Ga-transferrin complex. This provides another advantage of DFO, in that the leaching of certain metals to transferrin is eliminated.
Since DFO has no ionizable groups, its affinity for metals is not sensitive to low pH, which is yet another advantage over DTPA as a chelating agent.
Accordingly, the present invention provides stable conjugates of DFO and biotin for use in radioimmunoimaging and radioimmunotherapy. As discussed hereinabove, the compounds of the present invention have been developed to overcome the deficiencies of the commercially available biotin derivative, DTPA-biotin, which is a conjugate of DTPA and biocytin. Biocytin is a lysine conjugate of biotin which is the main form of biotin in foodstuffs, and is useful for synthesis of biotin derivatives since it is readily conjugated due to the availability of a primary amine. Biocytin is commercially available in its N-protected form as succinimidyl-6-(biotinamide)hexanoate (NHS-LC-biotin). However, it has been discovered in accordance with the present invention that a conjugate of biocytin covalently bonded to DFO (defero-desaminolysyl-biotin, DLB) is unstable, as demonstrated hereinbelow, and therefore unsuitable for use in two-step imaging and therapy. The DFO-biotin conjugate is rapidly degraded to biotin and desaminolysyldeferoxamine. The site of cleavage mimics the site in biocytin at which digestion by biotinidase occurs. Biotinidase is an enzyme found in high concentrations in plasma, gut, liver and other tissues which catalyzes the hydrolysis of biocytin to biotin and free lysine as follows ##STR1##
Biotinidase also digests short biotinyl peptides. It has been demonstrated that biotinidase is not a general proteolytic enzyme, but rather has .specific structural requirements in the substrate for hydrolysis [Chauhan et al. (1986) J. Biol. Chem. 261, 4268]. Therefore, it has become a further object of the present invention to provide DFO-biotin conjugates which are not subject to rapid degradation by biotinidase.